Project Summary An unexpected response from a minority of cells can have a dramatic impact on the development, prognosis, and treatment of disease. For example, in the progression of cancer, a resistant minority of circulating tumor cells (CTCs) lead to the development of refractory metastases at relapse. We are only just beginning to uncover these phenomena and the underlying biological mechanisms because they are obscured at the bulk scale. Detection of a rare mutation can require the analysis of hundreds to thousands of individual cells. We have developed a dielectrophoresis (DEP)-based device with a high yield of selective single-cell capture in an array of microchambers arranged along the sides of branched microfluidic channels. An important point is that DEP-based selection is marker-independent, which critically, makes this approach especially relevant to the identification of CTCs in melanoma, for which there are no reliable biomarkers (cell surface antigens). We propose to integrate genetic assays into this platform for the identification of a mutation that is an indicator for a specific chemotherapeutic agent that is in widespread use. Specifically, we will create devices and methods to assess mutations by incorporating structures and custom materials that facilitate on-chip end-point polymerase chain reaction (PCR). An important feature of our technology is that the transfer of cells into reaction chambers (for genetic analysis) is accomplished using simple fluidic and electrokinetic components (no moving parts). This technological development is significant because 1) it addresses a need for integrated marker-free selection, isolation, and analysis of single cells, 2) the platform is sufficiently simple for broad application in clinical laboratories, thus enabling characterization of CTCs isolated from individual patients, and 3) it will enable future basic research into the molecular mechanisms of acquired resistance in melanoma. This innovative combination of a platform for marker-free selection with detection of mutations in melanoma will transform melanoma treatment and research because it will allow unparalleled access to information regarding the distribution of mutations related to susceptibility or resistance to therapeutic agents among individual CTCs.